BIOTECHNOLOGIA ACTA, V. 11, No 5, 2018

UDC 615.919+577.152.34 https://doi.org/10.15407/biotech11.05.042

AMPHIBIAN SKIN SECRETIONS: A POTENTIAL SOURCE OF PROTEOLYTIC

I. Nikolaieva1 Yu. Dudkina1 1Taras Shevchenko National University of Kyiv, Ukraine D. Oliinyk1 O. Oskyrko1 2Schmalhausen Institute of Zoology O. Marushchak2 of the National Academy of Sciences of Ukraine, Kyiv T. Halenova1 O. Savchuk1

E-mail: [email protected] Received 18.06.2018 Revised 01.08.2018 Accepted 30.10.2018

The aim of the work was to study the protein content and proteolytic activity of the skin glands secretions of 10 the most common types of amphibians on the territory of Ukraine such as B. bombina, B. variegata, B. bufo, B. viridis, R. temporaria, P. ridibundus, P. esculentus, P. fuscus, S. salamandra, as well as the hybrid of B. bombina and B. variegata species. It was shown that the skin secretions of the studied amphibians contained a wide range of proteins with a molecular weight in the range from 8 to 150 kDa. By electrophoresis using gelatin, fibrinogen and collagen as substrates, it was found that they contained proteinases that differ in substrate specificity. It was revealed that the skin glands secretions of B. bombina, S. salamander species, as well as the hybrid of B. bombina and B. variegata species were characterized by the increased protein content with gelatinase and collagen activity.

Key words: amphibians, skin gland secretions, proteolytic activity.

Amphibians are recognized as a rich indicate that the amphibian glandular source of bioactive compounds with valuable secretion is a rich source of various molecules biotechnology potential and according to their with cardiotonic [2, 3,], antidiabetic [4], availability and accessibility they are supposed immunomodulatory [5], antimicrobial [6, 7] to be a superior raw material. The molecules and antiviral [8, 9] activities. It has also been derived from amphibian skin secretions established that they have sedative [10] and have various activities that determine their analgesic effects [11]. Moreover, considering profound applications in chemical and medical the availability and accessibility of some species industries. In this regard, one of the main of amphibians the further study of the effects objectives of modern biotechnology is the of the compounds from their skin secretions is search for new potential biologically active very relevant. compounds of natural origin. Since their In most of the cases the wide range of the effectiveness and safety cannot be compared effects of the components of skin secretions with chemically synthesized compounds, are associated with proteolytic activities. which characterized by a great number of side Proteolytic enzymes are capable of hydrolyzing effects and unpredictable actions, they might peptide bonds in proteins and have great have beneficial application in the science and medical and pharmaceutical importance due industry. to their key role in biological processes, such The promising raw materials that contain as: in digestion of food proteins, protein different bioactive molecules are various turnover, cell division, blood-clotting cascade, plants, marine invertebrates and reptiles. signal transduction, processing of polypeptide In the last two decades scientists drew their hormones, apoptosis and the life-cycle of attention to amphibians as potential objects several disease-causing organisms including for biochemical studies and industrial the replication of retroviruses [12]. Alongside purposes [1]. The great number of researches are extensively applied enzymes in

42 Experimental articles several sectors of industry and biotechnology Samples preparation and numerous research applications require The samples of lyophilized skin secretions the use of them [1]. were resuspended in Tris-buffered saline Although the territory of Ukraine is (TBS), pH 7.4 (30 mg of dried material/ml) dwelled by numerous amphibians, there and centrifuged at 7000 g for 15 min. Protein are a few studies concerning the nature concentration in supernatant was assayed and properties of the biologically active by Bradford method [14], using bovine compounds of their skin secretion. So, serum albumin as a standard. Samples for the purpose of this work is to examine sodium dodecyl sulfate polyacrylamaide the presence of proteolytic enzymes in gel electrophoresis (SDS-PAGE) and for the skin secretions of the most common zymography assay were mixed in equal Ukrainian species of amphibians and to volumes with the standard SDS-PAGE sample evaluate the gelatinolytic, fibrinogenolytic buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and collagenolytic activities to create a 5% sucrose, and 0.002% bromophenol blue) background for further investigations of without heating. amphibian secretions and for studying of their pharmaceutical potency. Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE of crude skin secretions was Materials and Methods carried out using 4% (w/v) stacking gel and Collection of frog skin secretions 12% (w/v) separating gel [15]. SDS-PAGE There are a great number of ways of was performed using Mini-Protean Tetra collection of the crude skin secretions that System (Bio Rad, USA) at 19 mA for stacking are shown in the studies, but most of them and 36 mA for separating gels. The volume are inhumane. They usually include lethal of sample applied per line was 15 μl. The gels release of the venoms, when the skin of were stained with 2.5% coomassie brilliant animals that previously were subjected to blue R-250 in 10% (v/v) ethanol, 10% (v/v) decapitation, is removed, dried and ground to acetic acid, 15% (v/v) isopropanol and the powder consistency for further use. Another background of the gel was destained with lethal variant is to place a frog in a flask 7% (v/v) acetic acid for 30 min. Apparent with anhydrous ether, which stimulates the molecular weights of proteins were estimated secretion of the skin poison that is washed using protein calibration mixture (Bio Rad, from the surface of the animal with deionized USA) containing myosin, -galactosidase, water. There are also some modern non- phosphorylase b, serum albumin, ovalbumin lethal methods, which include the usage of (Mr 97; 66; 45; 31; 21; 14 kDa). an electric current that causes synchronous release of toxic secretions from the glands or Zymography the stimulation of poison release by chemical Zymography was done according to the injection [13]. In our research, we have used method Ostapchenko et al [16]. Separating gel safe methodological approach, which allows (12% w/v) was polymerized in the presence us to use amphibians the unlimited number of of gelatin (1 mg/ml), fibrinogen (1 mg/ml) or times. collagen (1 mg/ml). The volume of samples Adult pubescent specimens (both sexes) of applied to the gel was 15 μl per line. After Bombina bombina (n = 20), Bombina variegata electrophoresis, the gels were incubated for (n = 20), Bufo bufo (n = 15), Bufotes viridis 30 min at room temperature on a rotary shaker (n = 10), Rana temporaria (n = 10), Pelophylax in 2.5% Triton X-100. Then the gels were ridibundus (n = 8), Pelophylax esculentus washed with distilled water to remove Triton (n = 5), Pelobates fuscus (n = 7), Salamandra X-100 and incubated in 50 mM Tris-HCl (pH 7.4) salamandra (n = 2) and hybrid of Bombina at room temperature for 12 hours. The gels were bombina and Bombina variegata (n = 5) were stained with 2.5% coomassie brilliant blue collected outdoors in Kyiv region of Ukraine. R-250 in 10% (v/v) ethanol, 10% (v/v) acetic The crude skin secretions were obtained by acid, 15% (v/v) isopropanol for 30 min and then washing the skin with ultrapure water beyond destained. The digested bands were visualized mechanical stimulation of skin glands. Water as the nonstained regions of the zymogram gel. solutions of skin secretions of all species were centrifuged at 3000 rpm for 15 min to remove Calculation of the results debris. The supernatants were lyophilized TotalLab 2.04 program was used to (TestarLyoQuest) and kept at 4 C till use. analyze the resultant electrophorograms. The

43 BIOTECHNOLOGIA ACTA, V. 11, No 5, 2018 represented electrophorograms and zymograms that different representatives of one type are typical for the series of the repeated of species had similar protein composition. experiments (at least three in each series). Thus, five common proteins were observed on the electrophoretic profile of crude skin secretion of amphibian species of Bombina Results and Discussion family. The differences between these two Amphibians’ glandular skin secretions studied secretions were only in two proteins are a rich source of potent biologically active (31 and 102 kDa), which were present in crude compounds, many with high potential for skin secretion of B. variegata. Noteworthy therapeutic drug development [17]. While is that the protein composition of the voluminous researches concerning the hybrid of B. bombina and B. variegata, was chemical structure and properties of crude almost identical to B. variegata. The protein skin secretions have been made all over the composition of the representatives of the Bufo world [18], relatively little is known about the family had more pronounced differences. Even protein composition and biological activities though all proteins that were discovered within of glandular secretion of different families of this family had low molecular weight, their amphibians that represent the batrahofauna of amount varied. Therefore, in B. bufo crude Ukraine. skin secretion five proteins ranging from 29 Therefore, on the first stage of our work we to 72 kDa was identified and nine proteins wanted to get information about the protein ranging from 8 to 68 kDa was observed in composition of glandular skin secretions of the B. viridis skin venom. The protein composition most common Ukrainian species of amphibians. of two representatives of Pelophylax family To achieve this the SDS-PAGE analysis was was also similar, except two proteins with performed. The typical electrophorograms molecular weights 31 kDa and 149 kDa, which of crude skin secretions are shown on the were present in P. ridibundus. The largest Fig. 1. The results of electrophoretic protein number of proteins that were observed among separation revealed the presence of proteins the studied amphibian species was identified with molecular weights ranging from 6 to in R. temporaria. It was found eleven low 149 kDa. It indicates that the crude skin molecular proteins ranging from 11 to 64 kDa secretions of studied amphibian species are in its glandular skin secretion. Only three characterized by a wide range of proteins with proteins 18, 31 and 46 kDa were observed in different molecular weights and alongside P. fuscus. It was also identified six proteins confirm a diverse protein composition of all in glandular skin secretions of S. salamander studied secretions. ranging from 10 to 60 kDa. To define the exact molecular weights The variety of proteins with different of identified protein fractions the molecular weights that are present in the crude electrophorograms were analyzed using the skin secretions of studied amphibian species TotalLab 2.04 program (Table). It was shown indicates the presence of different molecules

М 1 2 3 4 5 М 6 7 8 9 10

Fig. 1. Typical electrophoregram of crude skin secretions of studied amphibian species: 1 — B. bombina; 2 — B. variegata; 3 — hybrid of B. bombina and B. variegata; 4 — B. bufo; 5 — B. viridis; 6 — R. temporaria; 7 — P. ridibundus; 8 — P. esculentus; 9 — P. fuscus; 10 — S. salamander; М — markers of molecular weight

44 Experimental articles

The molecular weights (MW) of proteins that are present in crude skin secretions of studied amphibian species Amphibian species MW, kDa Bombina bombina 7; 14; 20; 27; 42. Bombina variegata 7; 17; 22; 26; 31; 40; 102. Hybrid of B. bombina and B. variegata 6; 13; 19; 27; 29; 41; 99. Bufo bufo 29; 41; 48; 52; 72. Bufotes viridis 8; 18; 29; 32; 35; 40; 50; 56; 68. Rana temporaria 11; 17; 20; 22; 24; 28; 31; 35; 42; 51; 64. Pelophylax ridibundus 14; 22; 24; 31; 54; 66; 109; 149. Pelophylax esculentus 11; 18; 22; 54; 69; 115. Pelobates fuscus 18; 31; 46. Salamander salamander 10; 19; 26; 30; 38; 60. that could have biological significance skin secretions of B. bombina, B. variegata, and might be a source of different types of the hybrid of B. Bombina and B. variegata, enzymatic activities. The presence of proteins B. viridis, P. esculentus and S. Salamander with molecular weight that is lower than 100 had pronounced gelatinolytic activity. Most kDa especially binds attention, since it is of the light digested areas corresponds to known that most of the proteolytic enzymes fractions of proteins with molecular weight have molecular weight up to 100 kDa. up to 70 kDa. Whereas it was not observed One of the simplest and the most expressed gelatinolytic activity in the study sensitive visual methods of the detection of of the components of crude skin secretions of active proteases in the biological material other amphibian species. is zymography method [19]. In this Collagen and fibrinogen are substrates with methodological approach the gels polymerize high specificity and the small amount of enzymes in the presence of the corresponding substrate are capable to hydrolyse them. On the other hand, proteins. And using different polymerized using these substrates can help us to identify and substrates we can identify the presence or detail the proteolytic enzymes action. absence of proteolytic enzymes. While studying the zymograms of crude In this research gelatin, fibrinogen and skin secretions with fibrinogen as a substrate, collagen were used as substrates to evaluate the the total fibrinogenolytic activity was proteolytic potential of crude skin secretions of insignificant and the hydrolysis zone was studied amphibian species. Our aim was to test identified only in the regions that corresponds the substrate specificity of proteolytic enzymes to the fractions of proteins with high molecular and to identify the presence of gelatinolytic, weight. The light digested zones correspond to fibrinogenolytic and collagenolytic activities. the pronounced fibrinogenolytic activity and The typical zymograms of the detection were revealed on the zymograms of the crude of gelatinolytic, collagenolytic, and skin secretions of B. bombina, S. salamander fibrinogenolytic activities are shown on Fig. 2. and the hybrid of B. bombina and B. variegate. The appearance of the light digested zones of Generally, the true fibrinogenolytic hydrolysis was due to the manifestation of enzymes have a molecular weight in the range enzymatic activity and indicated the presence from 20 to 60 kDa, but the enzymes with both of active proteolytic enzymes with the substrate lower and higher molecular weights are also specificity in the studied amphibian crude skin known [20]. The presence of high molecular secretions. The active proteins trypsin (24 kDa) weight proteolytic enzymes with specificity to and plasmin (84 kDa) were used to identify fibrinogen might be due to the inclusion of the the approximate molecular weights of active enzymes in the complexes. compounds. According to the results of zymography of Gelatin is considered as a common substrate crude skin secretions using collagen as a substrate, which helps to study the overall proteolytic it was revealed the presence active hydrolytic activity and usually used to pre-evaluate the enzymes that are capable to cleave collagen. presence of active forms of enzymes. According Moreover, the clearly defined zones of hydrolysis to the results the components of the crude were not detected, since the collagenolytic activity

45 BIOTECHNOLOGIA ACTA, V. 11, No 5, 2018 with different degree of severity was noted enzymes are a promising material for the throughout the length of the tracks. development of more specialized drugs [22]. Enzymes with collagenolithic activity are Thus, considering the obtained results, very interesting as they have a lot of practical we can state that the crude skin secretions of application in medicine, biotechnology and studied amphibian species have a pronounced food industry. Based on collagenolithic activity with specificity to different enzymes several medicines have already been substrates. The components of crude skin developed to treat wounds, burns and other skin secretions of B. bufo and R. temporaria had the lesions [21]. The new types of collagenolytic least evident gelatinolytic, fibrinogenolytic

T P 1 2 3 4 5 T P 6 7 8 9 10

А

T P 1 2 3 4 5 T P 6 7 8 9 10

В

T 1 2 3 P 4 5 P 6 7 8 T 9 10

С

Fig. 2. Typical zymograms of crude skin secretions using gelatin (A), fibrinogen (B) and collagen (C) as substrates: 1 — B. bombina; 2 — B. variegata; 3 — hybrid of B. bombina and B. variegata; 4 — B. bufo; 5 — B. viridis; 6 — R. temporaria; 7 — P. ridibundus; 8 — P. esculentus; 9 — P. fuscus; 10 — S. salamander; T — trypsin (24 kDa) and P — plasmin (84 kDa)

46 Experimental articles and collagenolytic activities, whereas the crude coincided. This can be the evidence of the skin secretions of B. bombina, S. Salamander presence of enzymes that simultaneously and the hybrid of B. Bombina and B. variegata have two or three kinds of activities. Further characterized by the most expressive activities investigations concerning the identification and, what is worth noting, some zones of of other biologically active compounds in hydrolysis on the zymograms with gelatin, the skin secretions of amphibians and their fibrinogen and collagen used as substrates characterization are required.

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СЕКРЕТИ ШКІРНИХ ЗАЛОЗ АМФІБІЙ — СЕКРЕТЫ КОЖНЫХ ЖЕЛЕЗ АМФИБИЙ — ПОТЕНЦІЙНЕ ДЖЕРЕЛО ПОТЕНЦИАЛЬНЫЙ ИСТОЧНИК ПРОТЕОЛІТИЧНИХ ЕНЗИМІВ ПРОТЕОЛИТИЧЕСКИХ ЭНЗИМОВ

І. Ніколаєва1, Ю. Дудкіна1, И. Николаева1, Ю. Дудкина1, Д. Олійник1, О. Марущак2, Д. Олейник1, О. Марущак2, О. Оскирко1, Т. Галенова1, О. Оскирко1, Т. Галенова1, О. Савчук1 О. Савчук1

1Київський національний університет 1Киевский национальный университет імені Тараса Шевченка, Україна имени Тараса Шевченко, Украина 2Інститут зоології ім. І. І. Шмальгаузена 2Институт зоологии им. И. И. Шмальгаузена НАН України, Київ НАН Украины, Киев

E-mail: [email protected] E-mail: [email protected]

Метою роботи було вивчити вміст протеїнів Целью работы было изучить содержание та протеолітичну активність секретів шкірних протеинов и протеолитическую активность залоз 10 найпоширеніших на території Укра- секретов кожных желез 10 самых распростра- їни видів амфібій: B. bombina, B. variegata, ненных на территории Украины видов амфи- B. bufo, B. viridis, R. temporaria, P. ridibundus, бий: B. bombina, B. variegata, B. bufo, B. viridis, P. esculentus, P. fuscus, S. salamandra, а також R. temporaria, P. ridibundus, P. esculentus, гібриду видів B. bombina та B. variegata. Пока- P. fuscus, S. salamandra, а также гибрида видов зано, що секрети шкірних залоз досліджуваних B. bombina и B. variegata. Показано, что секре- видів містять широкий спектр протеїнів з мо- ты кожных желез исследуемых видов содержат лекулярною масою від 8 до 150 кДа. Методом широкий спектр протеинов с молекулярной ензимелектрофорезу з використанням жела- массой от 8 до 150 кДа. Методом энзимэлектро- тину, фібриногену та колагену як субстратів фореза с использованием желатина, фибрино- виявлено, що вони містять протеїнази, які гена и коллагена как субстратов обнаружено, відрізняються за субстратною специфічністю. что они содержат протеиназы, которые отли- Встановлено, що секрети шкірних залоз видів чаются по субстратной специфичности. Уста- B. bombina, S. salamander, а також гібриду ви- новлено, что секреты кожных желез видов дів B. bombina та B. variegata характеризуються B. bombina, S. salamander, а также гибрида ви- підвищеним вмістом протеїназ із желатиназ- дов B. bombina и B. variegata характеризуются ною та колагеназною активністю. повышенным содержанием протеиназ с жела- тиназной и коллагеназной активностью. Ключові слова: амфібії, секрети шкірних залоз, протеолітична активність. Ключевые слова: амфибии, секреты кожных желез, протеолитическая активность.

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